Fast evolving RNA viruses, such as rotavirus, influenza virus, human immunodeficiency virus, and zika virus, are a leading cause of death worldwide and represent a major challenge for global disease control. Despite their miniscule genomes, often comprised of only a few thousand nucleotides and a handful of genes, it remains exceedingly difficult to study the infection biology of RNA viruses via modern gene sequencing technologies. RNA viruses evolve quickly. So quickly that even at the level of the individual infected cell, RNA viruses do not occur as a single viral genotype but rather as a swarm of closely related genotypes. This genetic diversity is a key determinant of the capacity of RNA viruses to escape cellular immunity, evolve drug resistance and cause emergent disease. The natural immune response of the infected cell in turn is highly heterogeneous, and dependent on cell type and state. The extreme heterogeneity of RNA virus infections is difficult to survey with current molecular technologies which are largely limited to analyzing populations of infected cells. To overcome this limitation, we have recently created a single cell RNA sequencing technology that combines multiplexed amplicon sequencing with single cell transcriptional profiling: Droplet Assisted RNA Targeting by single-cell Sequencing (DART-seq). DART-seq implements a combination of droplet-microfluidics based co- encapsulation of single cells with barcoded primer beads and ultra-deep DNA sequencing. With DART-seq it is possible to catalog the diversity of viral genome sequences within single infected cells, and at the same time record the cellular response to viral infection. The cost per-cell of DART-seq is less than one dollar, and a single DART-seq assay can yield measurements across thousands of cells in a biological sample. In a proof-of-principle study, we have used DART-seq to profile viral-host interactions and viral genome dynamics in single cells infected with mammalian orthoreovirus (REOV) strain Type 3 Dearing (T3D). Here we propose to expand upon these initial technology development experiments to interrogate the infection biology of REOVs and other segmented RNA viruses including rotavirus, a common cause of gastroenteritis, and influenza A virus. We will furthermore investigate the utility of DART-seq as a tool to perform high-throughput screens of the relationship between viral genotypes and single-cell infectivity and the relationship between host cell type or state and infection permissivity. This innovative project introduces a high-throughput single-cell analysis technology with transformative potential for investigations of the infection biology of RNA viruses. These studies are highly translational: DART-seq can lead to novel diagnostic approaches and can find application as a read-out tool for gene therapies that employ viral vectors.